Could long-silent, virus-like genomic elements trigger autoimmunity?

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Note that this series of experiments suggests that increased expression of protein derived from virus-like genomic inclusions may predispose to interferon production and thus systemic lupus erythematosus.

Some of these elements appear to be upregulated in the setting of oxidative stress, suggesting a path forward for treatment.

The vast uncoding region of the human genome -- once dismissed as "junk DNA" -- may hold an answer to the enduring question of what triggers the process of autoimmunity in lupus, a new study suggests.

Researchers led by Mary K. Crow, MD, of the Hospital for Special Surgery in New York City have found that virus-like endogenous elements that were long ago incorporated into the genome but that have no role in encoding for proteins may have a very different role: that of erroneously upregulating interferon in certain genetically predisposed individuals, ultimately leading to disease.

"A potential role for virus infection in the pathogenesis of autoimmune disease has long been an attractive hypothesis, in part based on clinical manifestations that mimic acute or chronic virus infection. Although production of interferon-1 is a key component of antiviral host defense ... there are scant data to directly implicate an exogenous virus in the interferon-1 pathway activation observed in many lupus patients," she and her colleagues wrote in Arthritis & Rheumatology.

"It's intriguing to think that virus-derived elements in our own genome are either quiet and don't cause any trouble, or they get stirred up and contribute to disease," said Crow, who is physician-in-chief and chair of the department of medicine and who also holds the Benjamin M. Rosen Chair in Immunology and Inflammation Research.

Triggers of Autoimmunity

One of the persistent mysteries of many autoimmune diseases involves the question of the initiating factors in the complex processes involved in the failure of the immune system to accurately distinguish "self" from "non-self," leading to tissue destruction and organ damage.

Many genetic factors have been identified in recent years, with the opening of the human genome, and simple chance is also likely to play a part, Crow told MedPage Today. But the third element of the tripartite basis for autoimmune disease -- an endogenous or exogenous trigger -- has proven more difficult to identify, she said.

One exception has been the association of cigarette smoking as an important trigger in the development of rheumatoid arthritis. Not only has a clear association been demonstrated, but a plausible mechanism also has been established, with post-translational citrullination of proteins in the lung exposed to tobacco smoke. This process has been shown to lead to the development of systemic autoantibodies, and ultimately, synovitis and clinical joint disease.

But no such specific trigger had been found for systemic lupus erythematosus (SLE), a highly complex disease. Previous research has shown that stimulatory immune complexes can induce production of interferon-α through toll-like receptors and their effects on plasmacytoid dendritic cells; triggering factors that might spur interferon upregulation earlier, however, before the typical auto-antibodies can be detected, had not been identified.

Crow and her colleagues have spent the past 15 years exploring the possibility that the answers in lupus may lie in the noncoding regions of the genome, and specifically in retroelements known as long interspersed nuclear element-1 (LINE-1 or L1), and the findings may hold important implications for disease understanding and treatment.

Retroelements and LINE-1

"Virus-like repetitive elements comprise a high portion of the human genome (roughly 48%)," she and her co-authors wrote. "Those sequences originated from retroviruses that integrated into our genome more than 25 million years ago. We hypothesized that these virus-like genomic elements might represent an endogenous source of ligands for nucleic acid sensors, induce interferon-1, and promote a host microenvironment supportive of immune dysfunction, autoimmunity, and inflammation."

The L1 group of retroelements represents about 17% of the genome, with some half-million copies being present. Most of these inserts are incomplete, mutated, or truncated at the protein-coding open reading frame 5′ region, and are therefore unable to be transcripted into proteins. However, some are full-length and are capable of RNA transcription, and the resulting proteins can be inserted at new locations in the genome.

Throughout genomic evolution, these potentially disruptive insertions have been tightly regulated, primarily through epigenetic methylation of DNA, and alterations in methylation in the L1 promoter region could permit increased expression and immune dysfunction, according to Crow.

"Our genomes are packed with sequences derived from viruses that were inserted many thousands of years ago, and these virus-like sequences can move around, causing genetic mutations and contributing to the evolution of our genomes. We hypothesized that they sometimes generate virus-like RNA sequences that can be detected by the immune system," she said in a press release.

The L1-Interferon Study

To address the question of a possible role for L1 in lupus pathogenesis, she and her team therefore conducted a series of experiments described as "elegant" in an accompanying editorial by Andras Perl, MD, PhD, of the State University of New York, Upstate Medical University, College of Medicine, in Syracuse.

The researchers first obtained biopsy samples of kidney tissues from 24 patients with lupus nephritis, and salivary gland tissue from 31 patients with Sjögren's syndrome, another condition associated with high expression of interferon.

Using polymerase chain reaction, immunohistochemistry, and Western blot analysis, the team determined that L1 transcripts were elevated in the renal tissue of lupus patients compared with healthy controls, with the highest levels seen among those with class IV nephritis. Similar findings were seen for salivary gland samples from the patients with Sjögren's syndrome, with greater expression of full-length L1 transcripts.

Staining with an antibody to ORF1/p40 (the open reading frame that encodes a 40 kilodalton RNA protein) detected the L1 protein in both renal tubular cells from the lupus patients and ductal epithelial cells from the Sjögren's patients.

"These results demonstrate that L1 retroelements are transcribed, and at least one of the L1 proteins -- ORF1/p40 -- is expressed in organs targeted by the immune system in SLE and Sjögren's syndrome," the researchers stated.

Further experiments determined that this increased L1 expression was associated with reduced methylation at the promoter CpG sites in the 5′ untranslated region of the L1 element. Of 9 CpG promoter sites tested, significantly reduced methylation was seen in 5, with a mean correlation coefficient of -0.598. "Methylation is known to be a key mechanism for controlling the expression of the L1 elements," Crow explained.

She and her colleagues then looked at L1 expression to see how it related to levels of interferon-1, and found correlations between L1 mRNA expression and interferon-α2 in lupus kidney samples as well as in Sjögren's salivary gland samples. "Collectively, these results point to a potential functional relationship between L1 transcript expression and interferon-1 production in SLE and Sjögren's syndrome tissues," the researchers observed.

Implications for Treatment?

Perl noted that further investigations into L1 and its effects on immunostimulation may provide new insights into potential therapeutic interventions: "Interestingly, LINE-1 transcription via hypomethylation is readily induced by oxidative stress, and it responds to treatment with the antioxidant N-acetylcysteine ... which is a potentially safe intervention with promising clinical benefits for patients with SLE."

He said he based that observation on the results of a pilot study he led in which N-acetylcysteine was administered in daily doses of 1.2, 2.4, or 4.8 g or placebo for 3 months to 36 patients with lupus. The treatment was associated with significant decreases in two disease activity indices, as well as with increased apoptosis and reduced anti-DNA antibody production.

As for the question of whether blocking interferon signaling itself is also being addressed in clinical trials, Crow noted that at least three monoclonal antibodies to interferon-α have been evaluated. One of these, rontalizumab, was not found effective in a phase II study, while another, sifalimumab, met its primary endpoint but the effects were modest.

However, results with a monoclonal antibody known as anifrolumab, which blocks the interferon 1 receptor, were recently presented at the European Congress of Rheumatology, showing that 63% of patients receiving 300 mg of anifrolumab once monthly met the primary study endpoint after a year of treatment. "These are the best lupus data we've ever seen," presenter Richard Furie, MD, of Hofstra Northwell School of Medicine in Great Neck, N.Y., told MedPage Today at the meeting.

An Evolutionary Perspective

"I've been fascinated by the study of retrotransposon elements and their role in evolutionary diversity and how organisms changed over time," Crow said in the interview. She referred to the work of Nobel laureate Barbara McClintock, PhD, on the genetics of maize beginning in the 1940s, whom she said "viewed the activation of endogenous transposons as a genomic defense mechanism against potential environmental threats, contributing to genetic diversity and the possibility of an effective adaptation."

She wrote: "Cells must be prepared to respond to many sources of stress. Mishaps that affect the operation of a cell must be occurring continuously. Sensing these and instigating repair systems are essential." She had found in her maize plants "potentially transposable elements that are carried in a silent state in the maize genome. The mobility of these [then] activated elements allows them to enter different gene loci and to take over control of action of the gene."

McClintock, who died in 1992, predicted that in the future, "attention undoubtedly will be centered on the genome, with greater appreciation of its significance as a highly sensitive organ of the cell that monitors genomic activities and corrects common errors, senses unusual and unexpected events, and responds to them, often by restructuring the genome."

Echoing McClintock's observations about the ongoing modifications of the genome, Crow and colleagues concluded their report by stating, "In this context, we speculate that autoimmune disease is the price we pay for the genomic flexibility that permits adaptation and survival of our species."

The study was supported by the Arthritis Foundation, the National Institutes of Health, the Lupus Research Institute, the Alliance for Lupus Research, and the Mary Kirkland Center for Lupus Research.